Antitumor agent

ABSTRACT

An antitumor agent containing 2,7,11-cembratriene-4,6-diol as an active ingredient, and a process for treatment of tumors with the antitumor agent, are disclosed.

TECHNICAL FIELD

The present invention relates to an antitumor agent and a process fortreatment of tumors, using it.

BACKGROUND ART

Synthetic chemical materials and antibiotics have been employed as lowmolecular antitumor agents. However, in general, many of these materialsare highly toxic and can cause side effects.

The development of an antitumor agent that has low toxicity to normalcells and causes few side effects has therefore been considereddesirable.

DISCLOSURE OF INVENTION

It is an object of the present invention to provide an antitumor agentthat has superior antitumor effects and that substantially does not showany toxicity to normal cells or display any side effects.

The present invention provides an antitumor agent containing, as anactive ingredient, 2,7,11-cembratriene-4,6-diol represented by thefollowing formula: ##STR1##

BEST MODE OF CARRYING OUT THE INVENTION

The active ingredient of the antitumor agent according to the presentinvention is 2,7,11-cembratriene-4,6-diol (hereinafter referred to asCBT) represented by Formula [I] below: ##STR2## CBT has twodiastereoisomers, i.e., 1S,2E,4S,6R,7E,11E-2,7,11-cembratriene-4,6-diol(hereinafter referred to as α-CBT) represented by Formula [II] below,and 1S,2E,4R,6R,7E,11E-2,7,11-cembratriene-4,6-diol (hereinafterreferred to as β-CBT) represented by Formula [III] below. Since bothα-CBT and β-CBT show antitumor effects, as will be apparent in Exampleswhich follow, the antitumor agent, according to the present invention,may contain, as an active ingredient, at least one of α-CBT and β-CBT.In this specification and the appended claims, the terms"2,7,11-cembratriene-4,6-diol" and "CBT" mean α-CBT and/or β-CBT. CBT isoften represented in a duvatriene form. Accordingly,4,8,13-duvatriene-1,3-diols, represented by Formulas [IV] and [V] belowrespectively, are the same compounds as α-CBT and β-CBT. It is to beunderstood that the compounds represented in the duvatriene form areencompassed within the scope of the present invention. ##STR3##

It is known that CBT is contained in resinous materials on the surfaceof tobacco leaves. It is also known that CBT is associated with tobaccoquality and its flavor during smoking. No pharmacological properties ofCBT are known except that it has the effect of preventing the growth ofpowdery mildew on tobacco (Japanese Patent Disclosure No. 58-157704. Inthis Disclosure, CBT is represented in the duvatriene form). The presentinventors have found for the first time that CBT has an antitumoreffect, and the present invention was completed based on this finding.

CBT may be produced, for example, in the following manner: Fresh or drytobacco leaves are immersed in an organic solvent such as hexane, ether,chloroform, or dichloromethane for a short period of time (for example,10 seconds to 60 seconds). The immersing temperature is normally between10° C. and 30° C. The weight ratio of tobacco leaves to the organicsolvent is preferably from 2:1 to 1:1. The immersing procedure may berepeated two or more times. The immersion extracts resinous materialsfound on the surface of the tobacco leaves. This extract is recovered byfiltration and is then condensed by evaporating the organic solvent at areduced pressure and a temperature of 40° C. or lower. The extract isconveniently condensed to a volume of about 1/25 to 1/200 of theoriginal volume.

The condensate is shaken after the addition of a hexane/methanol/watersolvent mixture, to transfer CBT to the methanol/water layer. Themethanol/water layer is then condensed in the same manner as theextract. The condensate is purified by silica gel chromatography using ahexane/ethyl acetate mixture as an eluting solvent. The removal of thesolvent from the eluate at a reduced pressure and a temperature of 40°C. or lower leaves CBT (with a purity of about 80% or higher). The CBTcan then be directly used as the active ingredient of an antitumor agentaccording to the present invention.

The CBT thus obtained may be subjected to high-speed liquidchromatography or alumina column chromatography to separate α-CBT andβ-CBT. The elution can be conducted, for example, by means ofhigh-performance liquid chromatography with an acetonitrile/water(70/30) solvent mixture using μBondapak C18 (available from Waters Inc.)as a filler. Then, the α-CBT (retention time: 18 minutes) and β-CBT(retention time: 21 minutes) are eluted separately.

CBT is preferably administered orally, subcutaneously, intravenously, orlocally.

The form of administering agent can be any one of the following:powders, granules, tablets, capsules, injectable solutions, localagents, and so on, prepared in a conventional manner by formulation witha pharmacologically acceptable carrier (an excipient such as lactose orstarch, or a solvent such as olive oil, soybean oil, or ethanol, forexample).

The dose of CBT can be determined according to the patient's symptoms,and the normal dose for an adult is from 1 mg to 100 mg per kg of bodyweight per day. It can be administered in several installments.

According to the present invention, the antitumor effects of CBT, whenadministered in the form of an antitumor agent, are believed to accruefrom its promoting the return of precancer tissues back to normal cells,or its blocking the conversion of precancer cells to malignant cells,such as is already known from the actions of vitamin A acid and relatedmaterials in this regard, or from control over carcinogenesis.Accordingly, CBT exhibits antitumor effects on tissues in the process ofcancer formation. CBT is also believed to produce these antitumoreffects without inhibiting as much of an immunological reaction as iscaused by other anticancer therapies or radiotherapies available today.Also, CBT is believed not to exert any particular influence on thefunction of normal cells when it controls the proliferation of tumorcells.

EXAMPLE 1 Production of CBT

Ten kilograms of fresh tobacco leaves in the budforming stage wereimmersed in 5 liters of dichloromethane for 30 seconds, and this processwas repeated twice to extract resinous materials on the surface of theleaves. The materials which remained insoluble in the dichloromethaneextract were removed by filtration, and the extract was condensed at atemperature of 40° C. or lower and at a reduced pressure (20 Torr). Thecondensate (0.1 liter) was shaken after the addition of 500 ml ofmethanol, containing 20% by weight of water, and 500 ml of hexane. Theresultant methanol layer was recovered and condensed at a temperature of40° C. or lower and at a reduced pressure (20 Torr). The condensate (40ml) was then dissolved in 100 ml of a hexane/ethyl acetate (80/20)solvent mixture, and 50 grams of silica gel (Wako Gel C-200) were addedthereto. The mixture was then treated at a temperature of 40° C. orlower and at a reduced pressure (20 Torr) to remove the solvent, leavingthe silica gel in which crude CBT was adsorbed.

The silica gel thus obtained was then placed on silica gel (Wako GelC-200) filled in a column (inner diameter: 5 cm; height: 85 cm) andsubjected to elution with a hexane, and then with a hexane/ethyl acetate(70/30) solvent mixture. A total of 4 liters of eluate was recoveredfrom the hexane/ethyl acetate solvent mixture. This eluate was condensedat a temperature of 40° C. or lower and at a reduced pressure (20 Torr)to thoroughly remove the solvent, leaving 13 grams of CBT, with an 80%purity.

A 0.5 gram portion of CBT was dissolved in an acetonitrile/water (60/40)solvent mixture and was then subjected to elution with anacetonitrile/water (70/30) solvent mixture by means of high-speed liquidchromatography (Model LC-5A available from Shimazu Seisakusho) usingμBondapak C18 as a filler to separate α-CBT (retention time: 18 minutes)and β-CBT (retention time: 21 minutes). Six grams of barelycrystallizable α-CBT and 2 grams of crystalline β-CBT were thusobtained.

    ______________________________________                                        Analysis of α-CBT                                                       Melting point:  65-66° C.                                              Specific optical                                                                              [α].sub.D.sup.25 + 281.6° C. (chloroform)        rotation:                                                                     Infrared spectrum:                                                                            3300(S), 1665(W), 1345, 1190,                                                 1160, 1118, 1024, 995, 974(S),                                                954, 818 cm.sup.-1                                            Ultraviolet spectrum:                                                                         λ .sub.max.sup.ethanol - 220 nm                        .sup.1 H-NMR spectrum                                                                         δ .sub.TMS.sup.CDCl.sbsp.3 ppm;                         (500 MHz):      0.79(d, J = 6.7 Hz, 3H),                                                      0.83(d, J = 6.7 Hz, 3H)                                                       1.34(S, 3H),                                                                  1.52(d, J = 1.0 Hz, 3H)                                                       1.67(d, J = 1.3 Hz, 3H),                                                      4.48(ddd, J = 2.0, 8.7 Hz, 1H),                                               5.04(bt, J = 5.0 Hz, 1H),                                                     5.33(3H)                                                      Mass spectrum:  (70 eV) m/Z (%)                                                               43(100), 55(42), 81(69),                                                      95(30), 121(18), 189(4),                                                      245(4), 288(0.1), 306(M.sup.+, 0.1)                           Analysis of β-CBT                                                        Melting point:  127-127.5° C.                                          Specific optical                                                                              [α].sub.D.sup.25 + 162° (chloroform)             rotation:                                                                     Infrared spectrum:                                                                            3280(S), 1667(W), 1166, 1142,                                                 1117, 1095, 1083, 1035, 975(S),                                               946, 923, 889, 875 cm.sup.-1                                  Ultraviolet spectrum:                                                                         λ .sub.max.sup.ethanol = 220 nm                        .sup.1 H-NMR spectrum                                                                         δ .sub.TMS.sup.CDCl.sbsp.3 ppm,                         (500 MHz):      0.80(d,J = 6.7 Hz, 3H),                                                       0.83(d, J = 6.7 Hz, 3H)                                                       1.40(S, 3H), 1.51(bs, 3H),                                                    1.70(d, J = 1.0 Hz, 3H),                                                      1.86(dd, J = 9.0, 14.1 Hz, 1H),                                               2.05(dd, J = 1.3, 14.1 Hz, 1H),                                               4.81(ddd, J = 1.3, 9.0, 9.4 Hz,                                               1H)                                                                           5.00(bt, J = 5.4 Hz, 1H),                                                     5.22(dd, J = 9.0, 15.4 Hz, 1H),                                               5.26(bd, J = 9.4 Hz, 1H),                                                     5.40(d, J = 15.4 Hz, 1H)                                      Mass spectrum:  (70 eV) m/Z (%)                                                               43(100), 55(59), 81(95),                                                      95(50), 121(35), 177(5),                                                      245(8), 306(M.sup.+, 0.1)                                     ______________________________________                                    

EXAMPLE 2 Antitumor Effects of CBT

A group of 6-week-old female ICR-JCL mice was inoculated,intraperitoneally, with a suspension of Sarcoma 180 cells at a dose of0.25 ml per mouse, and proliferated cancer cells were removed one weekafter inoculation. The cancer cells were transplanted at a dose of 1×10⁶cells per mouse to another 7 groups (each group consisting of 10 mice)of female ICR-JCL mice by subcutaneous injection in the hollow of onefront leg. α-CBT and β-CBT prepared in Example 1 above were eachformulated into an antitumor agent by dissolving them in olive oil to aconcentration of 0.15 to 1.5% by weight. The antitumor agent wasadministered to the mice every second day for a total of ten doses,starting 24 hours after transplantation of the cancer cells. Theantitumor agent was administered to the first to sixth groups of mice inthe CBT concentrations per dose listed in Table 1 below, and the seventhgroup of mice, used as a control group, was administered only olive oilin a total amount of 6,700 mg/kg. Tumor tubercles formed were removed 24hours after the tenth dose, and the tumor control rate was calculated bythe following formula:

    {1-(B/A)}×100(%)

in which A is an average tumor weight in the control group, and B is anaverage tumor weight in each of the groups to which CBT wasadministered. The results are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Antitumor Effect of CBT on Sarcoma 180                                        Compound     α-CBT  β-CBT                                          ______________________________________                                        Dose (mg/kg) 10    50      100  10    50  100                                 Tumor Control                                                                              25    48       67  22    46   64                                 Rate (%)                                                                      ______________________________________                                    

As is apparent from Table 1, both α-CBT and β-CBT show dose-dependentantitumor effects on Sarcoma 180.

EXAMPLE 3 Effects on Control of Tumor Formation

The fur on the backs of 7-week-old female ICR-JCL mice was shaved toexpose the skin, and 0.1 ml of an acetone solution containing 100 μg ofdimethylbenzoanthracene (DMBA) was applied to the exposed site.Thereafter, 0.1 ml of an acetone solution containing 0.5 μg of12-O-tetradecanoylphorbol-13-acetate (TPA) was applied to the site at arate of twice a week for 25 weeks, starting 1 week after the applicationof DMBA. In order to investigate the effct of CBT on control of tumorformation, an acetone solution containing 1 mg of α-CBT or β-CBT wasapplied to the site 40 minutes prior to every TPA application. Theformation of tumors was observed after 25 weeks for each group of mice,including a control group to which only TPA but not CBT was applied.Each group consisted of 30 mice. The tumor formation control rate wascalibrated by the following formula:

    {1-(C/D}×100(%)

in which C is the number of tumors formed in the group of mice to whichonly TPA was applied, and D is the number of tumors formed in the groupof mice to which CBT was applied before the application of TPA. C and Dwere observed with the naked eye. The results are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                        Effect of CBT on Control of Tumor Formation by TPA                            Compound   Tumor Formation Control Rate (%)                                   ______________________________________                                        α-CBT                                                                              80                                                                 β-CBT 65                                                                 ______________________________________                                    

As is apparent from Table 2, α-CBT and β-CBT both showed remarkablecontrol effects on tumor formation.

EXAMPLE 4 Toxicity

(1) Acute toxicity tests

The acute toxicity of CBT was investigated by intraperitoneal or oraladministration thereof to 6-week-old ICR-JCL mice and Wistar JCL rats. Asolution of α-CBT or β-CBT in olive oil was intraperitoneally injected,or was administered to the animals through an oral probe. The number ofanimals tested for each method of administration was 10. Afteradministration, observation was continued while the animals were fednormally for 7 days. The animals were killed on the eighth day to allowmacroscopic observations, and LD₅₀ values were calculated according tothe Richfield-Wilcockson method.

The anatomical observations showed no abnormalities. The LD₅₀ valuesobtained are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        Acute Toxicity Tests (LD.sub.50 Values)                                                    LD.sub.50 (mg/kg)                                                                   Intraperitoneal                                                                           Oral                                           Animal                                                                              Compound     Administration                                                                            Administration                                 ______________________________________                                        Mouse α-CBT  3000        4200                                                 β-CBT   3000        4000                                           Rat   α-CBT  3200        4100                                                 β-CBT   3100        3800                                           ______________________________________                                    

As is apparent from Table 3, the LD₅₀ values of CBT in both the mice andrats were higher than 3 grams per kilogram. CBT was therefore found tohave a high degree of safety, since it belongs to the "very weaktoxicity" category according to the Lumis classification.

(2) Subacute toxicity tests

A solution of α-CBT or β-CBT was orally administered to 6-week-oldICR-JCL mice through a probe every day for 20 days in a row, in a doseof 100 mg/kg. Only olive oil was administered to each mouse in a controlgroup in a dose of 6,700 mg/kg. Each group consisted of 10 mice. Themice were killed 24 hours after the last dose to allow hematologicalexaminations, anatomical observations, weighing of major organs, andpathohistological examinations. The results are shown in Tables 4 and 5.

                  TABLE 4                                                         ______________________________________                                        Hematological Examinations                                                           Number of Number of  Amount Hem-  GOT                                  Com-   Leukocytes                                                                              Erythrocytes                                                                             of Hb  atocrit                                                                             (mV/                                 pound  (cells/mm)                                                                              (cells/mm) (g/dl) Value ml)                                  ______________________________________                                        α-CBT                                                                          6300      6.1 × 10.sup.6                                                                     14.7   35%   52                                   β-CBT                                                                           6350      6.0 × 10.sup.6                                                                     14.5   35%   51                                   Control                                                                              6300      6.0 × 10.sup.6                                                                     14.6   35%   53                                   ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        Weights of Major Organs (g)                                                                             Left   Right                                        Compound                                                                              Liver    Spleen   Kidney Kidney Heart                                 ______________________________________                                        α-CBT                                                                           1.47     0.10     0.18   0.19   1.21                                  β-CBT                                                                            1.48     0.11     0.18   0.19   1.20                                  Control 1.49     0.11     0.18   0.19   1.20                                  ______________________________________                                    

As shown in Tables 4 and 5, the administration of CBT did not cause anyabnormalities in the hematological profile or organ weights. Inaddition, no abnormalities were found in body weight increase, amount offeed intake, macroscopic anatomical observations, or pathohistologicalexaminations (by means of an optical microscope).

(3) Mutagenesis tests

The mutagenesis test was conducted according to the Ames method(Mutation Research, Vol. 113, 1983, p. 173) with the use of 4 strains ofSalmonella typhimurium: TA1535, TA1537, TA98, and TA100.

0.1 ml of a Salmonella suspension (number of bacteria: 2×10⁸), 0.5 ml ofan S9 mix (prepared by adding nicotine adenine dinucleotide, in anamount of 3.2%, to a supernatant obtained by centrifuging rat liverhomogenates at 9,000 G) or 0.5 ml of a phosphate buffer (pH 7.4), and0.1 ml of a dimethylsulfoxide solution of α-CBT or β-CBT were added, inthat order, to 2 ml of soft agar (containing 0.05 mM of histidine orbiotin) heated to 40° C. After stirring, the mixture was cast on agarplates. The agar plates were incubated at 37° C. for 2 days, andcolonies formed thereon were counted as the number of reverse mutants.

Tables 6 and 7 respectively show the results when S9 mix was added andwhen S9 mix was not added.

                  TABLE 6                                                         ______________________________________                                        Mutagenesis Tests (S9 mix added)                                              Amount       Number of Reverse Mutants                                        Compound                                                                              μg    TA1535    TA1537 TA98   TA100                                ______________________________________                                        α-CBT                                                                            0       23        7      45     129                                          100      20        8      46     129                                          500      23        7      43     125                                          1000     21        6      41     120                                  β-CBT                                                                             0       23        7      45     129                                          100      19        7      47     108                                          500      17        9      48     130                                          1000     18        8      43     126                                  ______________________________________                                    

                  TABLE 7                                                         ______________________________________                                        Mutagenesis Tests (No S9 mix added)                                           Amount       Number of Reverse Mutants                                        Compound                                                                              μg    TA1535    TA1537 TA98   TA100                                ______________________________________                                        α-CBT                                                                            0       15        9      47     100                                          100      16        7      45     101                                          500      16        8      50     108                                          1000     17        7      43      98                                  β-CBT                                                                             0       15        9      47     100                                          100      15        7      48     100                                          500      14        7      49     101                                          1000     16        7      49     103                                  ______________________________________                                    

Tables 6 and 7 demonstrate that α-CBT and β-CBT are highly safecompounds which substantially do not induce the reverse mutation ofSalmonella strains in either case of adding S9 mix or of not adding S9mix.

We claim:
 1. An antitumor agent comprising, as an active ingredient,2,7,11-cembratriene-4,6-diol represented by the formula: ##STR4## and apharmacologically acceptable carrier for said active ingredient, suchthat said active ingredient is provided to a subject in orally,subcutaneously, intravenously or locally administrable form, whereinsaid active ingredient is present in an amount sufficient to provide adose of 1 to 100 mg per kg of subject body weight per day.
 2. Anantitumor agent according to claim 1, in which the active ingredient is1S,2E,4S,6R,7E,11E-2,7,11-cembratriene-4,6,-diol.
 3. An antitumor agentaccording to claim 1, in which the active ingredient is1S,2E,4R,6R,7E,11E-2,7,11-cembratriene-4,6-diol.
 4. An antitumor agentaccording to claim 1, in which the active ingredient is a mixture of1S,2E,4S,6R,7E,11E-2,7,11-cembratriene-4,6-diol and1S,2E,4R,6R,7E-2,7,11-cembratriene-4,6-diol.
 5. Use, for treatment oftumors, of 2,7,11-cembratriene-4,6-diol represented by the formula:##STR5## in combination with a pharmacologically acceptable carrier,such that said active ingtredient is provided to a subject in orally,subcutaneously, intravenously or locally administrable form, whereinsaid active ingredient is used in an amount sufficient to provide a doseof 1 to 100 mg per kg of subject body weight per day.
 6. Use, accordingto claim 5, of 1S,2E,4S,6R,7E,11E-2,7,11-cembratriene-4,6-diol fortreatment of tumors.
 7. Use, according to claim 5, of1S,2E,4R,6R,7E,11E-2,7,11-cembratriene-4,6-diol for treatment of tumors.8. Use, for treatment of tumors according to claim 5, of a mixture of1S,2E,4S,6R,7E,11E-2,7,11-cembratriene-4,6-diol and1S,2E,4R,6R,7E,11E-2,7,11-cembratriene-4,6-diol.
 9. A process fortreatment of tumors in humans, comprising the step of administering to asubject an antitumor agent which contains, as an active ingredient, anantitumor-effective amount of 2,7,11-cembratriene-4,6-diol representedby the formula: ##STR6## and a pharmacologically acceptable carrier forsaid active ingredient, such that said active ingredient is provided inorally, subcutaneously, intravenously or locally administrable form. 10.A process according to claim 9, in which the active ingredient is1S,2E,4S,6R,-7E,11E-2,7,11-cembratriene-4,6-diol.
 11. A processaccording to claim 9, in which the active ingredient is1S,2E,4R,6R,7E,11E-2,7,11-cembratriene-4,6-diol.
 12. A process accordingto claim 9, in which the active ingredient is a mixture of1S,2E,4S,6R,7E,11E-2,7,11-cembratriene-4,6-diol and1S,2E,4R,6R,7E,11E-2,7,11-cembratriene-4,6-diol.
 13. A process accordingto claim 9, in which the active ingredient is administered at a dose of1 to 100 mg per kg of subject body weight per day.